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Cord Blood CD34+ Cells Differentiate into Dermal Dendritic Cells in Co-Culture with Cutaneous Fibroblasts or Stromal Cells  Zia U.A. Mollah, Setsuya Aiba,

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Presentation on theme: "Cord Blood CD34+ Cells Differentiate into Dermal Dendritic Cells in Co-Culture with Cutaneous Fibroblasts or Stromal Cells  Zia U.A. Mollah, Setsuya Aiba,"— Presentation transcript:

1 Cord Blood CD34+ Cells Differentiate into Dermal Dendritic Cells in Co-Culture with Cutaneous Fibroblasts or Stromal Cells  Zia U.A. Mollah, Setsuya Aiba, Hideaki Manome, Yumiko Yoshino, Hachiro Tagami  Journal of Investigative Dermatology  Volume 118, Issue 3, Pages (March 2002) DOI: /j x x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 The coculture with cutaneous fibroblast cell lines supports the growth of CD34+ hematopoietic stem cells and their differentiation into DC. Three ml of NDF-1, NDF-2, CF-1, CF-2 or CF-3 (1 × 105 cells per ml) were seeded in 60 × 15 mm style Petri dishes. When these cells became confluent, they were irradiated with 2000 rad. Afterwards, 3 × 104 cells per ml of cord blood CD34+ HPC were added into these fibroblast cultures. Cultures were kept at 37°C in a 5% CO2 humidified condition. Cells were recovered at 21–28 d of culture for analysis. As a control, CD34+ HPC were cultured with GM-CSF, TNF-α, and Flt3L. (a) The culture of CD34 + HPC without fibroblasts. (b) The coculture of CD34+ HPC with CF-2 (day 14). (c) The proliferating cells from the coculture of CD34 + HPC with CF-2 were harvested by gentle pipetting (day 20) and further cultured for 2 d without fibroblasts. (d) The proliferation of CD34+ HPC cultured in the upper chamber of a dividing chamber (day 20). (e) TNF-α stimulates DC derived from the coculture of CD34 + HPC with CF-2 to form cell aggregates. (f) TGF-β1 does not significantly alter the morphology of DC derived from the coculture of CD34+ HPC with CF-2. (g) The culture of CD34+ HPC with GM-CSF, TNF-α, and Flt3L (day 13). Journal of Investigative Dermatology  , DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 The coculture with CFCL induces the differentiation of CD34+ HPC into CD14+ monocytes and CD1a+ DC. CD34+ HPC were cultured alone in the complete medium for 7 d, with various CFCL (CF-1, CF-2, and NDF-1) without any exogenous cytokines for 21 d, or without CFCL in the presence of GM-CSF, TNF-α, and Flt3L for 10 d. After culture, the cells were recovered and analyzed by flow cytometry. These data are representative of three independent experiments. Journal of Investigative Dermatology  , DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 The coculture with CFCL induces the differentiation of CD34+ HPC into CD1a+ CD11b+ CD11c+ HLA-DR+ E-cad– DC. CD34+ HPC were cultured with CF-3 without any exogenous cytokines for 21 d, and then the cells were analyzed by flow cytometry. These data are representative of four independent experiments. Journal of Investigative Dermatology  , DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 DC differentiated from CD34+ HPC by CFCL do not express BG-associated antigen, Lag, but express FXIIIa. The cytospin slides for CD34+ HPC cocultured with NDF-1 were examined for the expression of BG-associated antigen, Lag (a) and FXIIIa (d). (b, e) demonstrate DC stained with relevant control antibodies for anti-Lag antibody and anti-FXIIIa antibody, respectively. (c) shows DC differentiated from CD34+ HPC cultured with a combination of GM-CSF, TNF-α, and Flt3L stained with anti-Lag antibody. Similar data were also obtained by the coculture with other CFCL. Journal of Investigative Dermatology  , DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 DC differentiated from CD34+ HPC by CFCL increase their CD86 expression by TNF-α. CD34+ HPC were harvested from the coculture after 21 d and then stimulated in the complete culture medium with 10 ng per ml TNF-α for 48 h. Their surface expression of CD83 and CD86 on CD1a+ cells was examined by flow cytometry. These data are representative of three independent experiments. Journal of Investigative Dermatology  , DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 DC differentiated from CD34+ HPC by CFCL are potent antigen-presenting cells in allogeneic mixed lymphocyte culture. Purified CD3+ T cells (2 × 105 cells per well) were cocultured in 96-well flat bottom microtiter plates with various numbers of CD34+ HPC cocultured with CF-3. After 4 d of culture at 37°C in a 5% CO2 humidified atmosphere, the cells were pulsed with 5 μCi per ml of [3H]thymidine during the last 16 h of culture. The mean ± SEM from three replicates were taken from each sample culture. To decrease the background proliferation, cultured CD34+ HPC were irradiated with 2000 rad. In some experiments, cultured CD34+ HPC were prestimulated with 10 ng per ml of LPS or TNF-α for 48 h before T cell stimulation. These data are representative of four independent experiments. Journal of Investigative Dermatology  , DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 7 Direct contact between CD34+ HPC and CFCL are required for their optimal differentiation into CD1a+ DC. Using dividing chambers separated with semipermeable membranes, we cultured CD34+ HPC in the upper chamber and CF-3 in the lower chamber. After 21 d of culture, the cells were recovered and analyzed by flow cytometry for the expression of CD1a and CD14. Similar data were also obtained by the coculture with other CFCL. These data are representative of four independent experiments. Journal of Investigative Dermatology  , DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

9 Figure 8 The production of GM-CSF by CFCL does not correlate with their ability to induce DC from CD34+ HPC. NDF-1, NDF-2, CF-1, CF-2, and CF-3 were cultured in complete medium. After the cells became confluent, the cultures were replaced with new complete medium and further cultured for 1 wk. The recovered supernatants were measured for GM-CSF and M-CSF by enzyme-linked immunosorbent assay. The levels of GM-CSF and M-CSF were calculated by using a standard curve obtained with recombinant GM-CSF (from 0 to 1000 pg per ml) and M-CSF (from 0 to 1000 pg per ml). Data are representative of two independent experiments, and reported as the mean ± SEM of triplicate determinations. Journal of Investigative Dermatology  , DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

10 Figure 9 Anti-M-CSF antibody partially suppresses the proliferation of CD34+ cells cocultured with CF-3. We cultured CD34+ HPC with CF-3 or M-CSF (100 ng per ml) in the presence or absence of anti-M-CSF antibody or irrelevant antibody for 21 d. For the coculture with CF-3, half the volume of the culture medium was replaced with the same volume of fresh medium every 7 d. For the culture with GM-CSF or M-CSF, we changed one-half the volume of culture medium every 4 d with fresh medium containing the same concentration of GM-CSF or M-CSF. In some of the cultures with M-CSF, we added anti-M-CSF antibody or irrelevant control antibody (10 μg per ml) every 4 d. After culture, we counted the number of recovered cells. Data are reported as mean ± SD of three independent experiments. *p < 0.05 by paired Student's t test. Journal of Investigative Dermatology  , DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

11 Figure 10 M-CSF is mainly responsible for the differentiation of CD34+ HPC into CD1a+ cells. We cultured CD34+ HPC with CF-3, M-CSF (100 ng per ml), GM-CSF (50 ng per ml) in the presence or absence of anti-M-CSF antibody or irrelevant antibody for 21 d. For the coculture with CF-3, 200–300 μl of fresh complete medium were added to the cultures every 6–7 d after culture. For the culture with M-CSF or GM-CSF, we changed one-half the volume of culture medium every 4 d with fresh medium containing the same concentration of M-CSF or GM-CSF, respectively. In some of the cultures with M-CSF, or GM-CSF, we added anti-M-CSF antibody, anti-GM-CSF antibody or irrelevant control antibody once every 4 d. After culture, we analyzed their surface phenotype by flow cytometry. Journal of Investigative Dermatology  , DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

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